Article
Reference
Control of eukaryotic phosphate homeostasis by inositol polyphosphate sensor domains
WILD, Rebekka, et al.
Abstract
Phosphorus is a macronutrient taken up by cells as inorganic phosphate (P(i)). How cells sense cellular P(i) levels is poorly characterized. Here, we report that SPX domains--which are found in eukaryotic phosphate transporters, signaling proteins, and inorganic polyphosphate polymerases--provide a basic binding surface for inositol polyphosphate signaling molecules (InsPs), the concentrations of which change in response to P(i) availability. Substitutions of critical binding surface residues impair InsP binding in vitro, inorganic polyphosphate synthesis in yeast, and P(i) transport in Arabidopsis In plants, InsPs trigger the association of SPX proteins with transcription factors to regulate P(i) starvation responses. We propose that InsPs communicate cytosolic P(i) levels to SPX domains and enable them to interact with a multitude of proteins to regulate P(i) uptake, transport, and storage in fungi, plants, and animals.
WILD, Rebekka, et al. Control of eukaryotic phosphate homeostasis by inositol polyphosphate sensor domains. Science, 2016, vol. 352, no. 6288, p. 986-990
DOI : 10.1126/science.aad9858 PMID : 27080106
Available at:
http://archive-ouverte.unige.ch/unige:85715
Disclaimer: layout of this document may differ from the published version.
1 / 1
!"# !#$
%&'%! !&()!$*#"!+,' )!'!
+%,-+
& " .! !-/!!-01!.!-" .! !-2#-!
#&34 5
01!.!'-2#-!/&34 5
01!" ,! .! !-2#-!/&34 5
01!.!'-,6" !0# !1 .! !-7 -5
$01!9'-"'-2#-!:7'&34 5
(, '9! /!!-!,! 9 .! !-2#-9! /!!
25;55
* !<9'-% /32#-=-5
+9!1!!>-#51!? 5'@5"5A' 5'!'!?5'@,5)5A 5-? 5'@%5,5A
''!!B -!'-5
'''!C#!!'3!5 1!'-1!!'%%%&!
1! !!!5'##!31 '
$@(DDA>ED(EF!,-FF(3'0< F5(G5ED$D5
!"# !#$
%&'%! !&()!$*#"!+,' )!'!+
%,-+
& " .! !-/!!-01!.!-" .! !-2#-!
#&34 5
01!.!'-2#-!/&34 5
01!" ,! .! !-2#-!/&34 5
01!.!'-,6" !0# !1 .! !-7 -5
$01!9'-"'-2#-!:7'&34 5
(, '9! /!!-!,! 9 .! !-2#-9! /!!
25;55
* !<9'-% /32#-=-5 +9!1!!>-#51!? 5'@5"5A' 5'!'!?5'@,5)5A 5-? 5'@%5,5A
''!!B -!'-5
"'!1'!!1- !1'!1'@"A5)!3 " # 1!! -'45)31!'&"C!3''
! -! 1'!1' 1! 1! ! 1! -1'!1' 1! -1!#!!! 1! -1'!1' !
@ "A 3'! !! ' 1! ! " # -5 &! ! 1 "!1! -1'!1'-'-
" 1!%!15 1 "'!!!&"C1!3' 1!!! " #!1!51!1!' "!
-!! " # !&"C! '!3' !1!!
"11!!1 5@D3!A
%!#1!! !3 ! 1'!1' 1#!! 1! -1'!1'5@E'A
"11!! --!11!- 1!!&"C!!!3!5' !@$FEDA !'G!"1!'!1! -1'!1'@1! -"A1! -
! 1!'1!@9A" 1!!!1
!1!@=5&A5,!'&"C!1 '"61!
1 ' 1! 1- " 5' " 1! "'!E* "'!HD
#'&"C!3''-&1 1!'!!3 "1 @=5&A5 1 &"C ! <&")%I &%% <G !I&"<G&I @")A 1!!3''"#!61!" !!
5=!!!&"C!F")!1 61#'1!!!
1!!''61!!#!"!3'
!! 5" -1!!'!!!&"C1!3'")'&"C
!- " # 5
!#&"C!-!!!"311'!!
61 1 1 ' &"C ! @=5 &A5 ' 1- !&"C&@*EA!'-9!1 6@A55D J ! ! @ &A5 & ! ' &"C ! ! ' -!1'!1'!@EA!''1'!1'1!C"@"!!1 ! -!1!# !1!LD*A3! #5H$J5J! ! 1# -5''' '-!'&"C!> !!!3! !
@MEDJA' NN!- !# '@=5%.A5'!' 3! 9 ' N$N(!!'!!!' 6 @=5%.A5 ) 6N(!1#!!!!@=5.&A5'G!'
!! !' '1!-NN# O1!O' 6N11
!@=5%.A5&"C!'! '!! !-3'1!!!3
!@A5
!!# 'G!'&"C' @=59A5
&# # /- -)C" ! ' N N 1 &"C B 1!!'!!!' @=590&A@A5% !
!!1!' !&"C)C" @=50A5&<3''-
!!1 !"!!-'!!/-"'% !' 6
N - - /-( ! N - /-( ! N @'!1' # ".9A
&"C)C"@=50A5!3'&<#!&"C!!
-!@=5&A'' !! '1#! -"@ A5
G, ! ! 3 -1 &"C& &"C)C" # ; ! " ! M $ D , 1# -@=5I&A5,!!".9&"C&"! !
!'G N' 6@PGA!#'- !! -@=5IA5' - ! ' PG ' !# ' ! &"C ! - '!1''G,!61'&"C!
!!!! '1! 1! -#! #'N' 6@=5%I&
&A5 !! 1!@A3'&"C&!3"
@=5 I &A5 % ! ' &"C ! - ! !# - 'B!" !!!@$- ;&9L/-&*/-&D
/-&A!! ';&9 -!% '!!"@=50I&A5
/ '1-!1'!1'@""A!' -3'&"C&@=5I&A5'
&"C!'! 1!# -' !3'1'!1'!
'!3 1- #-!" 5
!!!'1! !-!&"C!3 -41! -"-'
! -#! 3'' -4-'&"C!9!1 6 -"
# 50 !!' "( ;3''1!!! 1-!1'!1'@"" "A ! 91! -"!@=5&$A@FA5-'4$ "*
;!1!!' 9 -41! -"-'!!
!#DD,@=5%&$A5 !"&<#9! - ! !!
@=5%A5&"C&!'&"C!$ "* "(3'M$DF$DD!! - 3'>!'!--@=5.&&( &A5&"C&
3$ "*Q "( "$ "! "!B -'! ! 9 -41! -"-'@=5&(&* &A5
! &"C!!4$ "* "(3' "(
9#-!#DDR,5' "( !B- 4!61
! - # ! $ "* ! 3'' 3 ! ! - -'4 !3 B5 9!1 6!&"C9 &"C9 # ' "( 3''
## '-!!!@=59=&E &A5' '' 1 -!'1 '!! !-! @A5'9 '6 1'!1'!1! "( '!'".91!!1-&< '
&"C)C"@=509=&EA5".9;&9!'!'&"C "
@=59=A5%! '-!!3' "( '-'G !!1 -'!!' 6N@=5=A5.! " -' 4'&"CN' '1!5
&!!!# "( $ "* !&"C&
@=5%.&H &A59!1!!&"C& &"C& -
! ' !!1! -"-'-$ "*3' !!/-(&Q/-H&!%
!# -#1! -"1!!@=59A5'91! -"-'!! -&"C
! "! 41#!@A5
'%!1")<&")%I@")<A1!!'! !!''C"" 61!
!! "1!!'!!!''!!"#!1!@A5 "
' &"C%")< !1 ' !!
1'!-1 @A 3''%")<!5;(S% !! 1 1
!3'3 -1%")<!3' '".9;&9PG$'!3
!3'@=50&DA5".9;&91 '#- 1'!-1'' !! -'".9B!! &"C!!!
%")<@=50A5'#!! ! "''!"%")<1 "
'!!5 ' PG$ 1'!-1 ' 1! ! ' 6 N &"C !
!'11!'! "!!! '@=50A5 &'!!"
!".9;&9PG$1 3!1!3 -1 !1 '"61!!'6- !1!'@=5IA5I61!!"
#!31 '!!!".9;&9PG$ !#
63'!1!!!1 @=5=A5' !
!&"C%")<'1"61!'"#! '"5
" 63 !!113' !-!&"C)C"
' "!! !"1!!#!#! !@=5&A@A5
!&"C "(!1 6! !! !!!- 1! @=5 9=A 3'' - # !1! ! " ! -
!#!'1 T!1! @A !!' - 1!1 6 "'!EDF
"'!E$'!"'!E @A5&"C1!!3''<")1!
!'1!$,"@A5!'<&"C<")$ "*
!! !3 D U, @=5 %A5 G! 3 !- 611!""""""!%"!><&"CQ<")1!! !@=5.
&A5 "( !3'; !M$DU,3'$ "* !3' M* R,-5 !!
&"C&'<&"CF<")!1 6''1!'1-!1'!1'!1 '1!-&"C! # "!!@=5..
&A@A5 "(31'<&"C".91 '' !! "5= -3<&"C!! !
!<")13'! ! !$ "*5!#!!!
>&"C!F1!!!1 63'!!!!M$U,3''
! "!!@=5.A5' "1!!'1!!
1 &"C1!3''1!!5#''"!!'1 -!! -!6DDR, "!" '! 11'-! ! !1
&"C1!@A5'3! 61 3'-%!1 "$3''!
'! "( "" " @A'#'' "!' '!3!#"
#!1!@A5
< - !1' !' - ! 1 ' &"C !
! 1! ' ' ! ! 1! ! "
! @A51!'&"C! "($ "*3'
# !#!'"" "-' # ! >
='"" "$ "*! "(#'-9!1 6@=5%A5&! - 1! -" !11! !!' "(1! 1!1! -"
- ;3''"" " @ A5''1!
'1!!1!1 &"C!'!31!"" "
@=5A5= - "* # "#!-3' "( # !
@A @ &A5 "" "-' ' " -!&"C!"
!! '!3' !1!' "'!!
-! @A51 '''' -1 - !3&"C!! "Q"" "!3''! !#- 1!
$
#! 1'-! ! 1! @A5 =' - 3 B ! ' 1
! 5<3!!3!1#!1 -1 "Q"" "
-! !!#! !1!&"C!5'" 1'3- 31!1!'-1!# !1'!1'-!11 5
(
!%
5 ,5/I5<V&'& 1'!1'#!5&'EFE*
@HH(A5
5 559'!&5 5/& G3!&"'!1'%# -" 5 !
"#(&E$FD(@DA5
5 05&!#'1!!'&"C!!1!1'!1' '!!5$%"&')*EFE$@DA5
5 )595)7 .5"!,5& &595:=5,5=!'&"C!
!'- !3-1'!1'1!"'!HD 1!#-5'()!'+
DDFDDE@DDHA5
$5 05)I544!!5,0! 9!"W!95&!# 5"!
!'4!!'%!1")<#! #1'!1' !
!'6- 5"# 5',EEHFHD@DDA5
(5 5/#%#! 1'!1'1! !1'!1''!!%!15
"$# #* ''&I($*FE@D$A5
*5 05!#5!'"59',5&!5/5. !1'!1'61!-'
!#1!C"4!5 !*E((FE*@DA5
E5 ,5)!'!#9 -!!!-!1! -1'!1'1! -5 5*&,$F$(@DDHA5
H5 G5<3#=! !!"'!E1'!!"'!E$11!'
!1'3-!" &'!-#5( '-HH*FDD
@HH$A5
D5 .5&5"5&5&!G5)59'G <IG/ , % <G%0%"% <G
")<&")%I!'"'!1'1!"!0!' !!
%!1"'!1')!!5"# 5&($F(@DA5
5 ;50#9'4!! -!%!13''&"C!
# '#!1 ! !1'!1'!#!5"#+-,H($FH*$
@DDEA5
5 &5#'IC&0!!")<"1'1!!&'!!!"'!1' 0-#!!!&'!!& 5"#"&'.+E$FDD@D(A5
5 0505-!%5)54#5,5.5,! I5;5<V&'"!#=
&3'!"'!1'1!&5#5( 5&.DD$FD@DD*A5
5 5!#%!#,.1!!#! #1'!1'#!
!'# 1 5,-'-F@DDA5
$5 ,5 5"#&"C1'!1'1'!!")<&")%I&%% <G I&"<G&I%!15"$# #* '''H*F$@DA5
(5 :5#&"C&"C'1'!1'#!1!'!' 3'")1'!1'15"$# #* '''H$FE@DA5
*5 /5)! &5;YYY"5!Z%5&' &'1!3' 0 /#55.#5&,*EDF*E@DDEA5
E5 %5/!# !!#! ! -!# -!!1! -1'!1'
!1! -1'!1'5+ &/(H((FH*@DA5
H5 &5.5&' !! 1-!1'!1'>'-!-1'!1'[ !/-.DF(
@D$A5
D5 95%)5!'!,5&'35;!&5)'" 1%E1;1
*
4-#! #!! 1-!1'!1'-' !1'!1' ! 1! -1'!1' !&'!-#5+ &/+$*F$
@DD$A5
5 ,5%5/!;5,5=!95&5%" '!! !-!' -! !50'1-'**F*(@DDA5
5 G5<350"5<5.!3G3!1!!-!1'!1' ! 1! -1'!1'! &'!-## -!61! -5 ( 5''DHF@DDDA5
5 %5/#,!C"1- !!3' 1'!1'61!5$#,,.$*HFE@D$A5
5 /5.5&'#!11!-!! 1'!1'1!9< %:!
1!5$#5,(/DDFD$@DDA5
$5 5&5/&5, 505!I5;5<V&' !!9- 90;90; '!
9!1 6- !! "-!1'!1'55*'(DHF@DD*A5
(5 \5/##&"CG# - "'!1'& )!!'!' !3'")5"# 5&($E(FH*@DA5
*5 5"#"'!1'@"A#!!'-!! "!!"6'
!'!!1 1 >##!" !- '- 1'!1'!" !5"#"&'-'((F($*@DDHA5
E5 ,50#3!!! '61'!1'#!! 1-!1'!1'-' 1 5"#+/+(F($@DA5
H5 5&#!" 55.&559'!5%5=-505!!!1'-
%!1'!'1!!!! 1! -1'!1'5"$# #
* '+&(F(*@DD$A5
E
01'5.Y '!' 13' -5"' !1 ! G%<!EE5 %!!!!'#1!3''
"!0.3'!$ @&"C,&!%A$ \@&"C,&! .A$ @&"C&!A$ )@&"C)C"&<A$ @&"C9 "(A$ "@&"C9 "(A5 '&!3'!! !!1-" 61!-5'1!T3 -!'I!1'9! 'I!12!O&#'=3!
"! @="QDD*DA Q I9 % 5 DE$( ' I!1 ,! .! !-
<!@I,.<A! #!"!@!,5)5A'&3G! =DD%
EHDD%$HHHE@!5"5A&G="!!'1""DD"$*(D*@!)555A!I9 5$E&G=$E@!%5,5A50!6131!
"C "C !'&3/'&!" &' 0H
!'I!1&-'!!!= -! =5&11 !!
5
H
Fig. 1. SPX helical bundles provide a positively charged ligand binding surface. (A) Ribbon diagram of apo SPXScVtc4 with core helices in blue and surrounding helices colored from N- to C- terminus in yellow to red. (B) Ribbon diagrams of the reductively methylated SPXScVtc4(left), SPXCtGde1 (middle) and of SPXHsXPR1 (right) shown in two orientations (colors as in A). (C) Electrostatic surface potential of the SPX domains from (B) reveals the SPX basic surface cluster. (D) A sulfate ion is bound to the SPXHsXPR1basic surface cluster. Interacting residues are shown in bonds representation (PBC, in bold; KSC; in italics), a distant Lys residue (SC) is shown alongside. Residue numbers correspond to SPXHsXPR1 (SPXScVtc2 in brackets). (E) Table summaries for NMR titrations of wild-type and mutant SPXScVtc2and SPXHsXPR1with different candidate ligands.
A D
N
1 3
A
N
C
1 2
3
4
6 5
D
N
N C
C
N
C
N
C
N C
N C 180º
C C
B
E
Protein Ligand Kd [mM]
SPXScVtc2 wt Pi 4.8 0.6
SPXScVtc2 wt SO42- 7.1 0.3 PPi
SPXScVtc2 wt 0.53 0.13
SPXScVtc2 Y22F Pi 47.1 22.5 SPXScVtc2 K26A Pi 19.5 1.7 SPXScVtc2 K131A Pi 9.1 1.0 SPXScVtc2 PBC Pi 93.1 38.4 SPXScVtc2 KSC Pi 6.1 2.4
SPXScVtc2 SC Pi 7.5 1.6
SPXScVtc2 N14 Pi 45.3 3.1 SPXScVtc2 meth Pi 60.5 18.4
SPXHsXPR1 wt Pi 22.8 0.3
E
Y22 K158(127) K26
K161(130)
K162(131) K165(134)
N 1
2 3
44
SC K77(71)
2
3 4
10
N Y22
K26 K30
K125 K128
K129 K132
K133
22 33
4 5
1 2 3
5
N Y22
K26 K30
K143 K147 K146
K150
22
3
4
C C N
55
1 2
Y22
22
3 4
N
C
2
M1 NNH3+
O
N N N N N NHHHHHHHHHHH
K2 HN
N N NHHHH F3
Y22
K26 K30
2
22
1
2 44
5
C D
E
F
Protein Ligand Kd [M]
SPXScVtc2 * SPXScVtc2 * SPXScVtc2 SPXCtVtc4 SPXCtGde1
0.05 ± 0.002 0.04 ± 0.001 0.44 ± 0.19 0.07 ± 0.02 0.28 ± 0.02 5-InsP7
InsP6 InsP6 InsP6 InsP6
B A
0.25 SO4
2-
10-9 10-8 10-7 10-6 10-5 10-4 10-3 10-2 10-1 5-InsP7
InsP6
Pi
0.0 0.5 1.0 1.5
0.75 1.25
Ligand concentration [M]
synthesized polyP [nmol/g]
K147 K143
K150 K146
Fig. 2. SPX domains sense InsP signaling molecules.(A) VTC-dependent polyP synthesis by isolated yeast vacuoles in response to increasing concentrations of externally supplied Pi, SO42-, InsP6 and 5- InsP7. (B) Binding affinities of different SPX domains vs. 5-InsP7 and InsP6 by isothermal titration calorimetry and microscale thermophoresis (indicated by an*) (± fitting errors). (C) Ribbon diagram of SPXCtGde1in complex with InsP6 (in bonds representation, colors as in Fig. 1A) and including a 2Fo– Fc
omit electron density map contoured at 1.5 . (D) Close-up view of the SPXCtGde1 with InsP6 – protein interactions depicted as dotted lines. (E) Ribbon diagram and close up view of the SPXCtVtc4 – InsP6
complex. (F) Rotated view of the SPXCtVtc4 complex highlighting the interactions of InsP6with main- chain atoms of helix 1.
11
wt (PHO1 wild-type)
PBC (Y23F/K27A/K319A)
KSC (K315A/K318A/K322A)
SC (K136A) N15 pho1 - empty vector
D
0246810Pi content [mol Pi /g FW]
WT PBC KSC SC N15 pho1
WT PBC KSC SC N15 pho1 SPX3/ACT2 0.000.040.080.12
WT PBC KSC SC N15 pho1
MGD3/ACT2
WT PBC KSC SC N15 pho1 IPS/ACT2 02468100246810
E
F B
semi-conserved lysine structural control (SC) lysine surface cluster (KSC) phosphate binding cluster (PBC) ScVtc2 Y22 K26K30 K71 K127 K130K131 K134 ScVtc3 Y22 K26K30 K70 K126 K129K130 K133 ScVtc4 Y22 K26K30 K66 K129 K132K133 K136 CtVtc4 Y22 K26K30 K76 K143 K146K147 K150 CtGde1 Y22 K26K30 K65 K125 K128K129 K132 OsSPX4 Y25 K29K33 R85 K151 K154K155 K158 AtPho1 Y23 K27K31K136K315 K318K319 K322 HsXPR1 Y22 K26K30 K77 K158 K161K162 K165
A
0 0.5 1.0 1.5 2.0
-20 -15 -10 -5 0
0 500 1000 1500 2000 2500 3000 3500 4000
0 0.5 1.0 1.5 2.0
Corrected Heat Rate [μJ/s]kJ/mol of Injectant
Time [s]
Ratio
SPXScVtc2 KSC Kd = 60.6 ± 11.8 M SPXScVtc2 PBC Kd = 21.0 ± 4.0 M SPXScVtc2 wt Kd = 0.44 ± 0.18 M
C
synthesized polyP [nmol/g]
wt/wt Y22F/Y22F
K26A/K26A K126A/K129A
K130A/K133A K133A/K136A
time [min] time [min]
no ligand 0.5 M 5-InsP7
0 5 10 15 20 0 5 10 15 20
0.0 0.2 0.4 0.6 0.8
0.0 0.2 0.4 0.6 0.8
Fig. 3. Mutations in the SPX InsP-binding site affect polyP synthesis in yeast and plant Pi
transport and Pi starvation signaling. (A) Isothermal titration calorimetry of wild-type and of PBC and KSC mutant SPXScVtc2 vs. InsP6 (± fitting errors). (B) Table summaries of the conserved surface cluster residues in the different SPX-domain containing proteins used in this study. (C) PolyP synthesis by isolated vacuoles carrying VTC complexes with corresponding substitutions in the SPX basic surface clusters of subunits Vtc3 and Vtc4 and in the presence or absence of 0.5 M 5-InsP7. (D) Shoot phenotypes of 28-d-old T2 pho1-2 transgenic lines, carrying promPHO1::PHO1:GFP (labeled wild- type) or its mutant variants. (E) Shoot Pi content of the transgenic lines from D. (F) Normalized relative expression of selected Pi-starvation induced genes in the different pho1-2 complementation lines.
12
protein ligand Kd [μM]
OsSPX4/OsPHR2 OsSPX4/OsPHR2
OsSPX4/OsPHR2 OsSPX4/OsPHR2 OsPHR2 OsPHR2/5-InsP7
OsSPX4-PBC/OsPHR2 OsSPX4/OsPHR2 OsSPX4/OsPHR2 OsSPX4/OsPHR2
7.0 ± 1.0 n. d.
49 ± 13 n. d.
n. d.
11.2 ± 3.3
n. d.
n. d.
n. d.
80
0.3 ± 0.1* 2.5 ± 0.4* 5-InsP7
PPi
InsP6 Pi OsSPX4 OsSPX4
InsP6 PPPi
ATP 1,3,4,5,6-InsP5
OsSPX4/OsPHR2 OsSPX4/OsPHR2
5-InsP7 InsP6
A
OsSPX4-HA OsSPX4-HA OsPHR2
OsPHR2 HA-beads
20M ligand
input M PD
+ - -
+ - -
+ + InsP7
+ + Pi
+ InsP7
-
* *
< <
*
<
Corr. Heat Rate [μJ/s]kJ/mol of Injectant
B Time [s]
-20 -15 -10 -5
0 500 1000 1500 2000 2500 3000 3500 4000
0 2 1 0
1.0
0.5 1.5 2.0 2.5
5-InsP7 vs. OsSPX4/OsPHR2 InsP6 vs. OsSPX4/OsPHR2 InsP5 vs. OsSPX4/OsPHR2 InsP6 vs. OsSPX4-PBC/OsPHR2
Fig. 4. InsPs trigger the association of plant SPX proteins with PHR transcription factors. (A)In vitro pull-down of full-length OsSPX4 containing a C-terminal HA-tag. Interaction with full-length untagged OsPHR2 in the presence of either 20 M 5-InsP7 or Pi, respectively. (B) Isothermal titration calorimetry of OsSPX4/OsPHR2 vs. different ligands (5-InsP7, black; InsP6, dark-blue; 1,3,4,5,6-InsP5, magenta; OsSPX4-PBC mutant vs. InsP6, light-blue) and including table summaries for different titrations (± fitting errors, n.d. no detectable binding). The * indicates experiments performed by microscale thermophoresis.
13
!" #$% &# ''(
( # "' (
)'* *#
*
%+
,-!./
0#--/
% -/
-
( '$%$/#1!
2 !3# 34%%5 45 26%%$/4-7/895 # %% 4/:;<(5 45 = !%'# '4%$5 ' 1!
;+ #0 ' 3 2$/ 4-7->85=
= :(-- 45 ! ' 4( !!5 # 2$/ 6 2$: 4 -7-8:5 22- 4-7:9>5 2$/ 4-!-?,5 2- 4-!-8/5= %'4!5'# ! ;+ #0!#@2/ @:='!
' 1! 8+!
# # A B = 'C#= + AD:-4E,5&D#' 2$/ 6 = 4501+
=#= # -.1/ #
=#=@E;99F9; ,>G% = -;G += =9,H!E!# 4&%5-;
= '# :9 /.99+# /G= =A !3C#0 =!
4.9%I>8.991 :H! JH!K9-L 4'I'5&(D-#:.99%1 4A$5:
& 455 = 0+!,4('5D = # >999+# /G-% = .D% 1:M 4 5== =.
'4$5.$ 4:99B:@/IB:@/
>8:H!5.$# 4.9%I>8-1 : :
H!5=:$2 # # = :.9 3 89 ' 1! ;+ #=' # %$
-)-99 -; /G% # ;+! ##%$ = ' C1:M %! #= ' 8+ ## #= @2/
@: 1:M # = 3 # 3!+ # 454:9I1 @
>9,991 9.%5-; /G = + >. :;I;9 4 5 = = !3C1: !89 G
$/2!%%(' ##
-.ND4/.#ID:9I1 @
>9:.91 -E%(9.4:! +54%55 -.ND 3 4-.D:@/9-I1 @>.5' 9/D ' = ' = -. L 4'I'5 # 3 C
# + # 4 A5 = #= -:
#ID-.41/5:@//L-9999-I1 @>. ! # :.L4'I'5A ,O !2&&I&&&=D#
4D5$#=3 ( 2$/ = ! ' 3 = :(-- ' 4-9#ID:9I1 @;.,99 1 9.%5%2! 3-?L4'I'5 ,,.99-41/5:@/9-I1 @;. = 3 #-.L4'I'5#!
:-, O ! &E:? 0 405 ,
0 1+ 22-4.#ID:9I1 @>9 :>.1 9.E%(5 -/41/5:@/-;L4'I'5#9-
%I8. = ## ,9 L4'I'5%22- 3 !
# :/,O 0 !&E:?(#&;+
= 2-4-,.#ID:9I1 @>9:99 1 9:.%-9&;5#= ## #:.L4'I'5 ,,.99-41/5:(9-A!%I;.Q #-?.O D 2&&& ! 3 #:.L 4'I'5 #% # $/ # +=&;)2$/ -9#ID:9I1 @>9 :991 9:.%-9&; 3-,L4'I'5,,.9;L 4'I'5 % .9 -. L 4'I'5 = 3 !(# :>.
O= # ! D !2&&&E # #=2E45
% $/ 2!%% 2! = ' # # (45 # '%%4EA!&E,#,5 --4EA!&E-3,5 ('22-2$/ 2-
= #' ' 2$/ R/ R. %
= ' #@@%45 %D AS%4 #D5( = 45 + 3 % - =
@$&%M 45 =# @$("4)II===' #5 = =# (A45 =45
/
0 2$: 22- = 1 +3 .!&> =
&; ' & # 3 01+-.1! 2$:= = AB # = -9#ID 14:.I1 @>9:.91 -E%(9.%5 .9TDE:@= /.9TD%U1-/ = 01 :.1 :@/I1 :@/ >9
= 9-..-9:9 /9 -99 = 1 :@/
&;= 999:.99>.9:9/ - '' # &;
### &; ' ##2%
= + ( = :?8 B ;993 A($&&&!;99%#
1 +
= '# #+ 0 # + !-.1!V JS!-.1K 2$:
# = 45 -!-.1!V =
=-:840-5-9:/40:5+ =,;.9340-5
>8-:340:58:/ == ! 9;
#= #%:-4A5= 3#:.;40-5
= = = ,--.
45= #C
=WX WX1 - -.1
'E 4B5=#
.
= JDK # JK +
+ =
!"#
-!1 = A/993 :?8B ' E =) 4X 5 4#QQQCC Q'
# Q # 5 # 45 4 35 # ( # = '# 4E,X>:;QE:@X/>?QE,@E X,,-QE@!;X:.95 ,-J-K!1 ,-!1 =
= -!# -#A-;:3:9:3 :?8B ' ( # = 45-,J-K!1 ==-!#A-9- -:.3 :?8B '(# = ' # 4E, X>>9Q E,@E X /?9Q E@!;X,?.5 =0# (%?.
ACDA 2V% 0# %V>99
% 0#-, 0#-/
%= 3 #!
( = =' 45
! "
-99#49:>-9C5 $ ;.;#4:/;?C5
!+#$#! 42!(5=' = 14:D5%= '14:D5 E04-D5%
:?9#4:/;?C5E&= # =
;
,-!1 ( 4-. 5 + = '= /:/#4:/;?C5A(4>9L=
= 5 9G% += ' %=
04# )@(@(I@:9)-5#,:.#%
49:./?,L5-14/993:?8BE,XI5)>,/!>-;4:95 .>-!..94/5./>!.:?4.5.:,!/?;4-.5/8;4%F--.?93 -5//.!/::4/5,;94%F-:93-5:8/!:;,4/5Q,-Y-Z1 4-;:3:?8BE,XI5)!-,?!::9!:.>!,--Q,-14-;:3:?8B E,XI5)!-9.!!->,45!-?,!!:/-45!:/-!!:8>45!:8?!!,,;45Q
4@:@()@Q8)-59.Q4&5 .:./1:1 :@:/; J4M:1 5:MK
;;-9;,8;;-9;,,
&'(
+ %-->#499?:-9C5= '14:
D5/8TD49,;>/9C5EAS= = 8/TD 49,>/9C5A%0(4#$)&A45 5 %
= -. % = %D(
8.TD49,>/9C5@ :.TD49,>
/9C5%0(= %+= -9 ' %= 14:D5;/#49-8 :9C5!3#$#! /,9TD49/.
19-8::9C5-*! 3= %= -.
=49[5# =
,-1( + = '=
,:#49-8:9C5A(4>9L== 51=
' = :@4'# /G5
% = + 48.#99.,.8L5%
!-8 # 4= I@,)-6-):'I'5- 14/993:?8BE,XI5)-9;-4EAS5>,:!>9/4:85;?84 :5.>?!/>,4,95/9,4EAS5,,-!:?>4EAS5:.84-5-?.4 EAS5->,!-984EAS5Q,-Y-Z14-;:3:?8BE,XI5)!-?/
>
!:;-!-9;84 % F-,;35!-:/94 % F-/935Q ,-14-;:3 :?8B E,XI5)!-,>!!::>45!::>!!,-;45!-9-9!!--9.45!-:/94%F -:; 3 -:; 35Q 4@: E)@Q 8)-5) 9,Q 4&5 ;9;9@:>>4!5-/:?-/?--/:?-.9?
&'(
% '= % 89 :4:!,5 / :4;!>5-.#4?.
T-9C5 .!!&.& =' + !A@
= 4/)--9D5 88#499?.--C51 @, -/#I 4-/, T-,C5= % += / :4/ 5 = 4::D5= %# = :.% = '# = ==
C = 3! 3
= I .!!&.' 4;:#8,>
T \ 88L + = F
>/99-Q ];/L4;9/T5-,1 MQF-9:.>8 =
= 5 -14.993:?8B +X I5)/8:4%F?83%F?83-5//84%F?.3%F?.3%F?.
3-5//.4%F?.3%F?.3%F?.3-5/:.4%F?.3%F?.
3-5/--4:5Q ,-Y-Z14-;:3:?8B+XI5,,>
:?;:-8!/?;!8,;4 % F-8:35Q 4&5 ;->@:>> 4:!5 ,;8?9;;,;8?9;.
"(
0' =#=-D"E ,9['#
' @E;99 98!-? ' = 45$
=A #A(
' ' = 45%= !99/#I'
8
= 4-9IB@;8-.9B9.
::995 # (%!# #4-(%!#:/9 9:.#I 5 :>[
9.N.!&>( 89N C=+=
-;9N4-9IB@;8-.9B:99-:
E%(9-.L4'I'5%2!-99 -.NE(&5 ?;!=
= =C -.
!E(&+= %( +&1&2 4 E'5#^+F/-.^F..94F.,95 :>[
% 3= ' =
!;94#&5 '+
N# ' % + # += =
# &+#= ,99 _# B # % = # ' 99:#I40#:(5999.#I40#>5(
-9 :>[ = = % -99=+=-99
= _;8=1 @ = # &; = -/.!&,-/.;!&/ -,/.;!&.=
")"*
&%+=# 1 &% 4%(&5
= 3 # &%4:9I1 @>9:991 9:.
%5 += :.[-9TD&; :>9 T := _/9T=! 2$:'%
&.&/&,1 1 #= # # :# :99T=!2$:& 2$/
=-9T'-99T&;02-
;9T= 3 # &% #,991 # /99T&; @: .9T @2/
?
4>.T5= -9TD&# 4.99T5(%4:
51 4:51 4.51 4.5=_' -.9( # # &%
= 3 # 1 ( 3 E ( = ' %# @2/@:= _#
@2/4.99T5 @:4.9T5 -99T.!&>&;1 1 = # &.
&/&,=
)+*
A# &; .!&> 2$: = .9 = = .9 1%(!@#
/8845-. #:.I1 @>9:.9 1 9-#IA(99.L%=!:9%+= #-9 ::999 +# ' ### + =
# = % += 1%
41 % %# 5 % = # 1%--.41 %%#5 =.I,9I. II'%DE=
= .9L% == ,9L %M%! # ,+= #!#C # .= 1# ! ## = '
% &; .!&>#@2/!@:+@:
= ' # ` D # B E!(D&&E 41 %%#5-99 @:= +=-99 @2/ #:9%I89-.9B 9:.%9-#IA( 99.L%=:9%+= # -9 ::999 +# + = # =
= '+ DE==
:9L % == .9LD# ! # -9
+-:LA #!%M%! # , += #!#C &; =
, -&-'+.
" = A"/>/: U': U', U'/
" A"/>/:':))DS:',)) 1%:'/)) 2=
A"/>/:'/)) 2 4 5 # # $%: $%,#= # 45!
$%, $%/ # # = # # ,9; ,9, ' E ## C' $, $/= ##=2 $%+
' ' ==
##$ > =/9!?9L'
=!$,I$/4=I=5% ' = $% '
/ 01$2
( # ## #@- 0
;+!&& #= E,: 45' # :-
@- =@-2 =
#&!0#%#=
( & # ,- . $,-9- 45%: # = # # ! #,9T#I#> = #= = I#-9I-/ # -.9Na6:6- '=
45D 3 @-!0=
# bD>99 4b 5= (
--
;,c E&_'= -:1(& #=#1&
& # = % 3@-+' = # ! # =!
# -.!:9 D !# # # C -L #
=+ #3:. /[+ # -9 >/,99-.91 .E%(.
%(-E%%:91 0 -c 4E%(
5 -9 ! = % =
#. ;999+# # = # #
;9 :-999+# 0# = E!( ( !$E0 4 5% %%(!04-).999 5 # ! &#! 4 3 A# S( -)>.99 5= #=A# 4(' S(5
# = # & #V %D& #0 + '( (/ 012 #C!%!>
##=! # ! = ' 1(=
V ' !%! = # #+,99.`
C 4 # D S(5 "A + 4(
A50 3 # ,3'#=
,!.=C+
!3
!==# !#@2/4-!,:95 # ! (! # T( ( # 4A5@ 89TD !( :T#@2//T#
!# ##@:4-!/:;5:9T# 4.!&> 5:9
%I89-.9B9.%9.L4'I'5&(D '- D += ,9 /[ #=A
= # = = =.+:99TD= # -:
4:9%I89-.9B9.%9.L4'I'5&(D
=:9T.!&> 5 >9TD 4?.[5 E = 3E(#:T#@2/!(
/T#@2/!( #
"#(
=#=45=
:/ ,9[ =.NI J-:!
,415K4;9I5%=#=-8!:9 ,9[ @E;99
9/!-9=# 4-899+#, 5
= == !
=.NI J-:!,415K @E;999.(-9 C
= =-9B:@/ : ,9[
! '%= : -9 '= = =%=
= =='#= !89[
+ -9B:@/ = #-9 = - 3 !&
=+ =@/ !+ #D 45% '&> &; = %&; &> = ## ' ,!
&; = C1141=# 1 5 ,!&> = #&;B- '45
-,
+
2
(" #+ 3 '!= &%
3$% % ( ++ & $% 31+
(# 3 @- +&
3.!&> $%"( 3 ( = (
+
,- 3$% %4 %45:,,/86;94:9-.5
,: B #+B3(D # 0 4 $%
;:/6;:.4:99.5
,, 1 =#b! D)II+#I-9-9,8I:99>:8>4:99>5 ,/ B %, -%6>?.68994-??,5
,. ( %, -'5;.86;>/4:99>5
,; B= , -24 -65:-:;6:-,:4:99/5 ,> &E ' #,5& ,>.6,8,4:99>5
,8 %E0E#(%, -&6?//6?/>4:99,5
,? 1(A E ( ( # , 6,47
-99,>6-99/-4:99-5
/9 0 ( @ 4 &7,9.6,:94-??;5 /- %EE B %4 #05&&:/,6:.?4:99.5 /: & '' ,-7'- '?;::6?;:;4:9-.5
/, d "E#f(( %$%#.9?,6.-9/
4:9-/5
// D '% h %, $%7:,;.6:,>:4:99;5 /. 8 %$?/>6?;:4:99/5
/; ES ( ( $&&/;:6/;?4:99,5 /> #(0A( %$6>,.6>/,4-??85
/8 A(0 97--.6--84-?;;5
/? (3'( # ( $:/-;6:/::4:99;5
.9 (3'(AA1@( 0 0 4 #%
6' >,68.4:9-95
.- B 4 %%:.>>6:;,>4-?8,5
.: % 5:, - - + -5;
-$< $, 4&A1,!?999.-!9>!9:9-/5
., ( %4 4:9-;5)-9-9>/I_--;>-/?9>
./ ' 1', -24 -64-:9/6-:-/4:9-,5
-/
Pi
A
Pho87
Pho90
Spl2
PM nucleus
cytoplasm
Pho81
Pho85 Pho80
PHO5 Pho4 Pho2
vacuole PiPiPiPiPi
PiPi PiPiPi
Vtc4
Vtc2/3 Vtc1
extracellular
SPX SPX
SPX
SPX
SPX
Pi Na+
Pi Na+
Pho89 SPX
Gde1
Pi
Pi Pi
Pi Pi Pi
Pi Pi Pi Pi
Pi
TTM TTM
vacuole
Spl2
C
PM extracellular
XPR1 Pi
Pi
cytoplasm SPX
Pi Pi Pi
B
SPX NLA PHO2
Ub
PHT1;1
PM
PHO1
cytoplasm
nucleus
Pi Pi Pi Pi Pi
Pi
SPX1
PSI PHR1
apoplast
Ub
SPX
SPX
Ub EM
Pi Pi Pi
SPXSPX-MFS
Fig. S1. SPX domain-containing proteins are involved in Pihomeostasis.Schematic overview of SPX-domain containing proteins:
The name SPX refers toSYG1 andPho81 from yeast, and mammalianXPR1, all of which contain an N-terminal SPX domain (http://www.ebi.ac.uk/interpro/entry/IPR004331). (A) Yeast: Piis taken up via high- (Phosphate metabolism 89, Pho89) and low- affinity phosphate transporters (Phosphate metabolism 87, Pho87;Phosphate metabolism 90, Pho90). Their SPX domains interact with the Spl2 protein (Suppressor of PhosphoLipase C 1 deletion). The cyclin-dependent kinase inhibitor Pho81 regulates transcription of phosphate-regulated genes (such as the PHO5 gene) via the cyclin-dependent kinases Pho80/Pho85 and the transcription factors Pho2 and Pho4. The glycerophosphodiesterase Gde1 liberates Pifrom glycerophosphocholine.TheVacuolar TransporterChaperone (VTC) complex generates polyP for Pistorage in vacuoles. TTM (TriphosphateTunnelMetalloenzyme). (B) Plants harbor SPX domains in Pi transporters such as PHO1 (Arabidopsis PHOSPHATE 1) and SPX-MFS (Major Facility Superfamily, http://www.ebi.ac.uk/interpro/entry/IPR011701), and E3 ubiquitin ligases (NLA, NITROGEN LIMITATION ADAPTATION), which themselves controlPHosphateTransporter (PHTs) protein levels. Plants also contain small, stand-alone SPX proteins, which interact with Pi-responsive PHOSPHATE STARVATION RESPONSE (PHR) transcription factors. Pho2: E2 conjugase. PSI:Phosphate-starvation induced genes. (C) The only SPX domain-containing protein in mammals is the phosphate exporterXenotropic andPolytropic retrovirusReceptor 1 (XPR1). PM (plasma membrane), EM (endomembrane).
15
α6
α5
α3
α2
α4
ScVtc2 1-182 M-LFGVKLANE---VYPPWKGSYINYEGLKKFLKEDSVKDGS---NDKKARWDDSDE-SKFVEELDKELEKVYGFQLKKYNNLMERLSHLEKQTDTEAAI- ScVtc3 1-182 M-LFGIKLAND---VYPPWKDSYIDYERLKKLLKESVIHDGR---S-SVDSWSERNE-SDFVEALDKELEKVYTFQISKYNAVLRKLDDLEENTKSAEKI- ScVtc4 1-178 M-KFGEHLSKS---LIRQYSYYYISYDDLKTELEDNLSKNNG---QWTQELE-TDFLESLEIELDKVYTFCKVKHSEVFRRVKEVQEQVQHTVRLL ScPho90 1-322 M-RFSHFLKYN---AVPEWQNHYMDYSELKNLIYTLQTDELQ---- ---DTY-DTFVGDLTAEKQKVDDFYKRTEAKFYDKFDALVKDLKKI-GVI CtVtc4 1-197 M-KFGQQLRSS---IIREYQWHYIDYDGLKADLKRASG--- PPRREWTEDDE-SRFVSKLEAELDKVHAKQQVKAMEISRRIAVSEREVQDVVGRL CtGde1 1-184 M-KFGKNLPRN---QVPEWAGSYINYKGLKKLVKAAAESAKD----GQP---VDL-AEFFFALDRNLEDVDSFYNKKFADACRRLKVLQDRYGTTPEVV OsSPX4 1-206 M-KFGKDFRSHLEETLPAWRDKYLAYKSLKKLIKNLPPDGDP-PPV ---ALG-NWFARVLDMELQKLNDFYIEREEWYVIRLQVLKERIERVKAK- AtSPX1 1-191 M-KFGKSLSNQIEQTLPEWQDKFLSYKELKKRLKLIGSKTAD-RPV SVGISKEE-INFIQLLEDELEKFNNFFVEKEEEYIIRLKEFRDRIAKAK--- AtPho1 1-366 MVKFSKELEAQ---LIPEWKEAFVNYCLLKKQIKKIKTSRKP-KPA QLFSEEDEVKVFFARLDEELNKVNQFHKPKETEFLERGEILKKQLETLAELK HsXPR1 1-207 M-KFAEHLSAH---ITPEWRKQYIQYEAFKDMLYSAQDQAPSV--- ----AKFE-EKFFQTCEKELAKINTFYSEKLAEAQRRFATLQNELQSSLDAQ
ScVtc2 1-182 KALD--A-DAFQRVLEELLSESTELDNFKRLNFTGFAKIVKKHDKLYPKYPSVKSL-LEVRLKELPSHS-E---EYSPLLYRISFLYNILRS--N-FNTAS ScVtc3 1-182 QKIN--S-EQFKNTLEECLDEAQRLDNFDRLNFTGFIKIVKKHDKLHPNYPSVKSL-LQVRLKELPFNNSE---EYSPLLYRISYLYEFLRS--N-YDHPN ScVtc4 1-178 D--F-EILEEELSDIIADVHDLAKFSRLNYTGFQKIIKKHDKKTGFIL--KPV-FQVRLDSKPFFKEN---Y-DELVVKISQLYDI---ARTSG ScPho90 1-322 EY KK-SLLKKSIVNLYIDLCQLKSFIELNRIGFAKITKKSDKVLHLNTRT--EL----IESEQFFKDTYAFQAETIELLNSKI----SQLV---TFYARIT CtVtc4 1-197 -Q ---MLLEEDLSDIIADVHDLAKFVQVNYTGFYKIIKKHDKMTGWRL--KPV-FDTRLKAKPFYKEN---Y-DASVVRLSKLYDLVRT--RGNPAKG CtGde1 1-184 ---A-EELMGALLELRSQLRKLQWFGEINRRGFIKITKKLDKKVPNTTTQ-HRYISTKVDPKPFAKDT---TVARILTEINRWISVLGDARNVEDNRS OsSPX4 1-206 KNGA -LEIRKAFVIIHGEMILLQTYSSLNFAGLVKILKKYDKRTGGLLS--LP-FTQRARHQPFFTTE---PLTRLVRECEANLELLFP--IEAEVLE AtSPX1 1-191 DSMEK-M-IKIRKEIVDFHGEMVLLENYSALNYTGLVKILKKYDKRTGDLMR--LP-FIQKVLQQPFYTTD---LLFKLVKESEAMLDQIFP--ANETESE AtPho1 1-366 QIL AEKKIRSAFVELYRGLGLLKTYSSLNMIAFTKIMKKFDKVAGQNAS--ST-YLKVVKRSQFISSD---KVVRLMDEVES----IFT--KHFANND HsXPR1 1-207 K --I-KDLKLAFSEFYLSLILLQNYQNLNFTGFRKILKKHDKILETSR--GADWRVAHVEVAPFYTCK---KINQLISETEAVV---TNELE
Y22K26 K71
K130 K127 K131
K134
α1
K30
A
protein/ residues/ mutation tag organism
ScPho81 1-199 His [N] S accharomyces cerevisiae P17442 -
ScPho81 1-199 Strep [C] Saccharomyces cerevisiae P17442 -
ScVtc2 1-157 His [C] Saccharomyces cerevisiae P43585 +/-
ScVtc2 1-177 His [N] Saccharomyces cerevisiae P43585 -
ScVtc2 1-182 His [C] Saccharomyces cerevisiae P43585 +
ScVtc2 15-182 His [C] Saccharomyces cerevisiae P43585 +
ScVtc2 1-182 Strep [C] Saccharomyces cerevisiae P43585 +/-
ScVtc2 1-553 E475N (catalytic dead) His [C] Saccharomyces cerevisiae P43585 +
ScVtc2 1-176 Macro-His [C] Saccharomyces cerevisiae P43585 -
ScVtc4 1-156 His [C] Saccharomyces cerevisiae P47075 -
ScVtc4 1-178 His [C] Saccharomyces cerevisiae P47075 +
ScVtc4 1-178 His-Strep-Thioredoxin [N] Saccharomyces cerevisiae P47075 +
ScVtc4 1-178 Macro-His [C] Saccharomyces cerevisiae P47075 +
ScVtc4 1-480 E426N (catalytic dead) His [C] Saccharomyces cerevisiae P47075 + ScVtc4 1-480 E426N (catalytic dead) Strep [C] Saccharomyces cerevisiae P47075 - ScVtc4 1-480 E426N (catalytic dead) His [N] Saccharomyces cerevisiae P47075 +
ScVtc4 1-480 His [N] Saccharomyces cerevisiae P47075 +
ScVtc4 1-480 His [C] Saccharomyces cerevisiae P47075 +
ScVtc4 189-480 His [N] Saccharomyces cerevisiae P47075 +
ScPho87 1-383 His [C] Saccharomyces cerevisiae P25360 +
ScPho90 1-335 His [C] Saccharomyces cerevisiae P39535 +
CtGde1 1-193 His [C] Chaetomium thermophilum G0RY29 +
CtVtc4 1-188 His [C] Chaetomium thermophilum G0SCH1 +
CaSPX1 1-189 His [C] Cicer arietinum XP_004494019.1* +/-
CaSPX1 1-189 His [N] Cicer arietinum XP_004494019.1* +/-
CaSPX1 1-189 His-Strep-Sumo [N] Cicer arietinum XP_004494019.1* +/-
TcSPX 1-188 His [C] Theobroma cacao gb|EOY25792.1** -
TcSPX 1-188 His-Strep-Sumo [N] Theobroma cacao gb|EOY25792.1** +/-
HsXRP1 1-207 His [C] Homo sapiens Q9UBH6 +
AtPho1 1-344 His [C] Arabidopsis thaliana Q8S403 -
AtPho1 1-344 His-Strep-Sumo [N] Arabidopsis thaliana Q8S403 -
AtPho1 1-364 His-Strep-Sumo [N] Arabidopsis thaliana Q8S403 -
AtPho1 1-386 His [C] Arabidopsis thaliana Q8S403 +
AtSPX1 1-159 His [C] Arabidopsis thaliana Q8LBH4 -
AtSPX1 1-170 His [C] Arabidopsis thaliana Q8LBH4 -
AtSPX1 1-256 His [C] Arabidopsis thaliana Q8LBH4 +/-
AtSPX1 1-256 His-Strep-Thioredoxin [N] Arabidopsis thaliana Q8LBH4 -
AtSPX1 1-256 His-Strep-GB1 [N] Arabidopsis thaliana Q8LBH4 -
AtSPX1 1-256 Macro-His [C] Arabidopsis thaliana Q8LBH4 +/-
AtSPX1 1-256 His-Strep-Sumo [N] Arabidopsis thaliana Q8LBH4 +/-
AtSPX3 1-248 Strep [C] Arabidopsis thaliana Q5PP62 -
His [C] Arabidopsis thaliana Q5PP62 -
His [C] Arabidopsis thaliana Q5PP62 -
Macro-His [C] Arabidopsis thaliana Q5PP62 -
OsSPX4 1-320 His-Strep-Sumo [N] Oryza sativa Q10B79 +/-
OsSPX4 1-320 His-Strep-Sumo [N] + HA [C] Oryza sativa Q10B79 +/-
N: N-terminal
C: C-terminal * NCBI identifier
Macro: histone macro H2A1.1 domain ** GenBank identifiers
GB1: protein G B1 domain
Uniprot
identifier express- able
AtSPX3 1-237 codon optE.coli AtSPX3 1-179 codon optE. coli AtSPX3 1-171 codon optE. coli
B
Fig. S2. A conserved set of lysine residues forms a basic surface ligand binding cluster in SPX domains.(A) Sequence alignment of different SPX domains including a secondary structure assignment for SPXScVtc4calculated with the program DSSP (51). Conserved amino acid residues are highlighted in green (phosphate binding cluster, PBC), blue (lysine surface cluster, KSC), orange (semi- conserved surface lysine), gray (structural control mutant, SC). (B) Overview of the different SPX domain expression constructs and their biochemical properties.
16
N
C C
N N
N
CSPX-ScVtc4
CSPX-ScVtc4-red. met.
CSPX-CtGde1 CSPX-HsXPR1
A B
Fig. S3. The C-terminal 6 helix adopts various conformations in different SPX domain structures and crystal forms. (A) Structural superposition of crystal forms A and B of SPXScVtc4crystallized in fusion with the catalytic TTM domain (r.m.s.d. is 1.6 Å comparing 170 corresponding Catoms). Shown are Ctraces colored according to Figure 1A. (B) Structural superpositions of SPXScVtc4form A (in light-blue) with ScVtc4 form C obtained by carrier driven crystallization (in orange, r.m.s.d. is 2.2 Å comparing 135 corresponding Catoms), with SPXCtGde1(in dark-blue; r.m.s.d. is 2.3 Å comparing 141 corresponding Catoms), and SPXHsXPR1(in red; r.m.s.d. is 1.6 Å comparing 147 corresponding Catoms), indicates that the C-terminal 6 helix can adopt different orientations.
17
Titration on SPXHsXPR1
0 20 40 60 80 100
0.0 0.2 0.4 0.6 0.8 1.0
Ligand concentration [mM]
Chemical Shift
InsP6 Pi
Titration on SPXScVtc2
0 5 10 15 20 25 30
0.0 0.2 0.4 0.6 0.8 1.0
Ligand concentration [mM]
Chemical Shift
Pi PPi InsP6 SO4
2-
Titration on SPXScVtc2
Chemical Shift
Pi concentration [mM]
0 20 40 60 80 100
0.0 0.2 0.4 0.6 0.8 1.0
wt meth Y22FK26A K131A
A
Pi concentration [mM]
Chemical Shift
0 20 40 60 80 100
0.0 0.2 0.4 0.6 0.8 1.0
wt PBCKSC SC N14
Titration on SPXScVtc2
-1
1-15 N [ppm]
H [ppm]
105
110
115
120
125
130
10 9 8 7
0 mM 0.5 mM 1.5 mM 5 mM 10 mM 20 mM 40 mM 100 mM
C
1-15 N [ppm]
105
110
115
120
125
130
10 9 8 7
0 mM 0.025 mM 0.075 mM 0.2 mM 0.4 mM 1 mM
-1
B
&''4(2)&&' @' 2$: !-.1!V1 4+5 4/5&; ! ##4&;!
5 4 &;5 =4.51 3 = # ##
# -)-#E =' 3=# 45
-8
Ipk1 Kcs1
5-InsP7 1-InsP7
1,5-InsP8
5PP-InsP 1,3,4,5,6-InsP5 4
InsP6 Arg82 1,4,5-InsP3
1,4,5,6-InsP4 Arg82 2 1 3 4
5 6
Kcs1
Kcs1 Vip1
Vip1
Fig. S5. Schematic representation of known inositol polyphosphate and -pyrophosphate synthesis pathways in yeast. (modified from (53).)
19
